Development of Novel Small Antitumor Compounds Inhibiting PD-1/PD-L1 Binding.

Background/aim: Anti-programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) antibody is a successful treatment for patients with solid cancers; however, there are several disadvantages that need to be resolved. Oral small molecule anti-PD-1/PD-L1 inhibitors have been developed and have good bioavailability.

Materials And Methods: Potent anti-PD-1/PD-L1 inhibitor candidates from the Shizuoka small compound library were screened and investigated for their antitumor activities in vitro and in vivo using a humanized mouse model.

Immunological and Genetic Characterization of Patients With Head and Neck Cancer who Developed Recurrence.

Background/aim: The recurrence rate of head and neck squamous cell carcinoma (HNSCC) remains high; thus the control of recurrence is a clinical problem to be challenged. To clarify the precise mechanism, specific immunological biomarkers responsible for recurrence were investigated.

Patients And Methods: The expression levels of immune response-associated and Shizuoka Cancer Center 820 cancer-associated genes, and genetic mutations from whole-exome sequencing were compared between HNSCC patients who developed recurrence (n=8) and HNSCC patients who did not develop recurrence (n=19) using a volcano plot analysis.

Characterization of the Immunological Status of Hypermutated Solid Tumors in the Cancer Genome Analysis Project HOPE.

Background: Many reports demonstrate that a high tumor mutation burden (TMB-H) is closely associated with good prognosis of cancer. However, specific studies investigating the association of various TMB statuses with overall survival in patients with solid tumors are scarce.

Patients And Methods: In the present study, we investigated the association of TMB status with overall survival in 5,072 patients with cancer from the HOPE project and clarified the specific mechanism responsible for the good prognosis of the TMB-H group.

Development of an Automatic Measurement Method for CD8 and PD-1 Positive T Cells Using Image Analysis Software.

Background/aim: With the progress in cancer immunotherapy using immune checkpoint blockade (ICB) therapy, histological observations of tumor-infiltrating lymphocyte (TIL) status are needed to evaluate the antitumor effect of ICB using imaging analysis software.

Materials And Methods: Formalin-fixed paraffin-embedded sections obtained from colorectal cancer and gastric cancer patients with more than 500 single nucleotide variants were stained with anti-CD8 and anti-PD-1 antibodies. Based on our own algorithm and imaging analysis software, an automatic TIL measurement method was established and compared to the manual counting methods.

Identification of tumor microenvironment-associated immunological genes as potent prognostic markers in the cancer genome analysis project HOPE.

Project High-tech Omics-based Patient Evaluation (HOPE), which used whole-exome sequencing and gene expression profiling, was launched in 2014. A total of ~2,000 patients were enrolled until March 2016, and the survival time was observed up to July 2019. In our previous study, a tumor microenvironment immune type classification based on the expression levels of the programmed death-ligand 1 (PD-L1) and CD8B genes was performed based on four types: A, adaptive immune resistance; B, intrinsic induction; C, immunological ignorance; and D, tolerance.

Alternative Evaluation of an ELISPOT Assay Using Cytokine Activity as a Novel Parameter.

Background/aim: The enzyme-linked immunospot (ELISPOT) assay is a well-established method used to evaluate the strength of T cell-mediated immune activity, and accepted as a standard functional immunological assay. Cytokine activity is a novel parameter reflecting spot size and intensity, which has not been used in ELISPOT assay before.

Materials And Methods: In the present study, from 113 ELISPOT assay data derived from previous clinical trials with dendritic cell vaccines, both spot number count and cytokine activity data for IFN-γ secretion were obtained using an ELISPOT reader.

Alpha-type-1 Polarized Dendritic Cell-based Vaccination in Newly Diagnosed High-grade Glioma: A Phase II Clinical Trial.

Background/aim: Glioblastoma multiforme (GBM) is an intractable tumor that has a very poor prognosis despite intensive treatment with temozolomide plus radiotherapy.

Patients And Methods: Sixteen newly diagnosed patients with high-grade gliomas were enrolled in a phase II study of the α-type-1 DC vaccine. Briefly, DCs obtained from the culture of enriched monocytes in the presence of a cytokine cocktail, were pulsed with a cocktail of 5 synthetic peptides and cryopreserved until injection into patients.

Effect of preoperative chemoradiotherapy on the immunological status of rectal cancer patients.

The aim of the study was to investigate the effect of chemo-radiation on the genetic and immunological status of rectal cancer patients who were treated with preoperative chemoradiotherapy (CRT). The expression of immune response-associated genes was compared between rectal cancer patients treated (n = 9) and not-treated (n = 10) with preoperative CRT using volcano plot analysis. Apoptosis and epithelial-to-mesenchymal transition (EMT) marker genes were analysed by quantitative PCR (qPCR).

Antitumor activity of the PD-1/PD-L1 binding inhibitor BMS-202 in the humanized MHC-double knockout NOG mouse.

Recently, the first series of small molecule inhibitors of PD-1/PD-L1 were reported by Bristol-Myers Squibb (BMS), which were developed using a homogeneous time-resolved fluorescence (HTRF)-based screening investigation of the PD-1/PD-L1 interaction. Additional crystallographic and biophysical studies showed that these compounds inhibited the interaction of PD-1/PD-L1 by inducing the dimerization of PD-L1, in which each dimer binds one molecule of the stabilizer at its interface. However, the immunological mechanism of the antitumor effect of these compounds remains to be elucidated.

Impact of combination therapy with anti-PD-1 blockade and a STAT3 inhibitor on the tumor-infiltrating lymphocyte status.

Recently, clinical studies using anti-immune checkpoint molecule antibodies have been successful in solid tumors, such as melanoma and non-small cell lung cancers. However, pancreatic cancers are still intractable and difficult to treat once recurrence or metastasis occurs; thus, novel combined use of immune checkpoint blockade (ICB) with molecular targeted drugs is considered a therapeutic option. Previously, we developed a novel humanized MHC-double knockout (dKO) NOG mouse model and demonstrated that an anti-PD-1 antibody or a STAT3 inhibitor showed anti-tumor effects through an immunological mechanism.

Identification of a neoantigen epitope in a melanoma patient with good response to anti-PD-1 antibody therapy.

Recent advances in next-generation sequencing have enabled rapid and efficient evaluation of the mutational landscape of cancers. As a result, many cancer-specific neoantigens, which can generate antitumor cytotoxic T-cells inside tumors, have been identified. Previously, we reported a metastatic melanoma case with high tumor mutation burden, who obtained complete remission after anti-PD-1 therapy and surgical resection.

Classification of tumor microenvironment immune types based on immune response-associated gene expression.

In 2014, the Shizuoka Cancer Center launched project High‑tech Omics‑based Patient Evaluation (HOPE), which features whole exome sequencing (WES) and gene expression profiling (GEP) of fresh surgical specimens from cancer patients. With the development of clinical trials of programmed death‑1 (PD‑1)/PD‑ligand 1 (PD‑L1) blockade, PD‑L1 expression and a high tumor mutation burden become possible biomarkers that could be used to predict immune responses. In this study, based on WES and GEP data from 1,734 tumors from the HOPE project, we established a tumor microenvironment (TME) immune‑type classification consisting of 4 types to evaluate the immunological status of cancer patients and analyze immunological pathways specific for immune types.

Immune response-associated gene profiling in Japanese melanoma patients using multi-omics analysis.

Project High-tech Omics-based Patient Evaluation (HOPE), including comprehensive whole-exome sequencing (WES) and gene expression profiling (GEP) using freshly resected tumor specimens, has been in progress since its implementation in 2014. Among a total of 1,685 cancer patients, 13 melanoma patients were registered in the HOPE Project and were characterized using multi-omics analyses. Among the 13 melanoma patients, 4 were deceased, and 9 were alive.

The anti-tumor activity of the STAT3 inhibitor STX-0119 occurs via promotion of tumor-infiltrating lymphocyte accumulation in temozolomide-resistant glioblastoma cell line.

STAT3 is considered to be a key molecule to mediating tumor-induced immunosuppression in various manners at tumor sites, by acting through immune-regulatory cytokines derived from the tumor cells. Specific anti-STAT3 inhibitors have been developed using nude mouse models transplanted with human tumor cells. However, mouse systems cannot accurately represent the human immune response induced by STAT3 inhibitors, and more humanized therapeutic model based on human immune cells and tumors are needed.

Melanoma patient response to nivolumab treatment for metastatic lung lesions: Multi-OMICS analysis in Project HOPE.

A 70-year-old woman was diagnosed with a malignant melanoma of the occipital skin which was resected; however, multiple lung metastases were detected. Nivolumab therapy was initiated and partial response was obtained. However, the patient was diagnosed with grade 2 interstitial pneumonitis.

Multi-omics Profiling of Patients with Melanoma Treated with Nivolumab in Project HOPE.

Background: Project HOPE (High-tech Omics-based Patient Evaluation) has been in progress since 2014 and uses whole-exome sequencing (WES) and gene expression profiling (GEP). Among a total of 1,685 patients with cancer, 13 with melanoma were registered and characterized using multi-omics analyses to investigate specific biomarkers in responders to programmed cell death-1 (PD-1) blockade.

Materials And Methods: The patients with melanoma comprised of six males and seven females, and their mean age was 68 years.

Combination of a STAT3 Inhibitor and an mTOR Inhibitor Against a Temozolomide-resistant Glioblastoma Cell Line.

Background: Temozolomide-resistant (TMZ-R) glioblastoma is very difficult to treat, and a novel approach to overcome resistance is needed.

Materials And Methods: The efficacy of a combination treatment of STAT3 inhibitor, STX-0119, with rapamycin was investigated against our established TMZ-resistant U87 cell line.

Results: The growth-inhibitory effect of the combination treatment was significant against the TMZ-R U87 cell line (IC: 78 μM for STX-0119, 30.

Unstable B7-H4 cell surface expression and T-cell redirection as a means of cancer therapy.

Tumor immune regulation has been demonstrated in clinical studies using antibodies targeted to the B7/CD28 family. B7 homolog 4 (B7-H4) negatively regulates immune responses and is overexpressed in many types of human cancer, indicating that B7-H4 may be a potential target of cancer therapy. B7-H4 expression is affected by the microenvironment, and its presence has been reported in cancer tissues and immune cells.

Immunological effects of the anti-programmed death-1 antibody on human peripheral blood mononuclear cells.

Immune checkpoint antibody-mediated blockade has gained attention as a new cancer immunotherapy strategy. Accumulating evidence suggests that this therapy imparts a survival benefit to metastatic melanoma and non-small cell lung cancer patients. A substantial amount of data on immune checkpoint antibodies has been collected from clinical trials; however, the direct effect of the antibodies on human peripheral blood mononuclear cells (PBMCs) has not been exclusively investigated.

Immune response-associated gene analysis of 1,000 cancer patients using whole-exome sequencing and gene expression profiling-Project HOPE.

Project HOPE (High-tech Omics-based Patient Evaluation) has been progressing since its implementation in 2014 using whole-exome sequencing (WES) and gene expression profiling (GEP). With the aim of evaluating immune status in cancer patients, a gene panel consisting of 164 immune response-associated genes (56 antigen-presenting cell and T-cell-associated genes, 34 cytokine- and metabolism-associated genes, 47 TNF and TNF receptor superfamily genes, and 27 regulatory T-cell-associated genes) was established, and its expression and mutation status were investigated using 1,000 cancer patient-derived tumors. Regarding WES, sequencing and variant calling were performed using the Ion Proton system.

Antitumor Effect of Programmed Death-1 (PD-1) Blockade in Humanized the NOG-MHC Double Knockout Mouse.

Purpose: Humanized mouse models using NOD/Shi-scid-IL2rγ (NOG) and NOD/LtSz-scid IL2rγ (NSG) mouse are associated with several limitations, such as long incubation time for stem cell engraftment and the development of xenograft versus host disease in mice injected with peripheral blood mononuclear cells (PBMCs). To solve problems, we used humanized major histocompatibility class I- and class II-deficient NOG mice (referred to as NOG-dKO) to evaluate the antitumor effect of anti-programmed death-1 (PD-1) antibody.

Experimental Design: Humanized NOG-dKO mice, in which human PBMCs and human lymphoma cell line SCC-3, or glioblastoma cell line U87 were transplanted, were used as an immunotherapy model to investigate the effect of anti-PD-1 antibody.